Characteristics of ChgH–GFP transgenic medaka lines,an in vivo estrogenic compound detection system

We previously reported the characteristics of a ChgH–GFP transgenic medaka line that indicates estrogenic compound pollution in environmental water by the green fluorescence of their liver. Recently, we established four more lines. In this study, the characteristics of the five transgenic medaka lines were investigated. The intensity of reporter gene expression varied among transgenic lines and generally correlated well with the amount of integrated transgene in each line. Line-specific ectopic expression was also observed. However, the sensitivity to 17-β estradiol did not differ among transgenic lines. Three transgenic lines are considered to be suitable as bio-indicators of estrogenic activity, due to the ease of observing green fluorescence in their livers. The transgenic lines can also detect the estrogenic activity of testosterone and 17-β trenbolone at the nominal concentration of 30 and 100 μg/l, respectively.     

Characteristics of ChgH-GFP transgenic medaka lines, an in vivo estrogenic compound detection system

We previously reported the characteristics of a ChgH-GFP transgenic medaka line that indicates estrogenic compound pollution in environmental water by the green fluorescence of their liver. Recently, we established four more lines. In this study, the characteristics of the five transgenic medaka lines were investigated. The intensity of reporter gene expression varied among transgenic lines and generally correlated well with the amount of integrated transgene in each line. Line-specific ectopic expression was also observed. However, the sensitivity to 17-beta estradiol did not differ among transgenic lines. Three transgenic lines are considered to be suitable as bio-indicators of estrogenic activity, due to the ease of observing green fluorescence in their livers. The transgenic lines can also detect the estrogenic activity of testosterone and 17-beta trenbolone at the nominal concentration of 30 and 100 microg/l, respectively.     

Alteration of gene expression profiles in the brain of Japanese medaka (Oryzias latipes) exposed to KC-400 or PCB126

Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals and possibly alter animal behavior. We previously reported that PCB-exposure induced abnormal schooling behavior in Japanese medaka (Oryzias latipes). This abnormal behavior might be caused by the functional alteration of central or terminal nervous system. To understand the mechanism(s) of behavioral change by PCB-exposure, we analyzed the gene expression profiles in the brain of medaka exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) or a PCB mixture (Kanechlor-400: KC-400) using a cDNA microarray that we constructed. Twelve FLF-II strain medaka (six individuals per treatment) were dietary exposed to PCB126 (0.01 μg/g b.w./day) or KC-400 (1 μg/g b.w./day) for three weeks. For the control, six fish were fed a control diet. After the exposure period, fish were dissected, and the brain samples were collected. The samples from control fish were pooled and used as a common reference in the microarray experiment. Microarray data were normalized by the LOWESS method, and we screened the genes whose expression levels were altered more than 1.5-fold. Gene expression profiling showed 97 down-regulated and 379 up-regulated genes in the brain of medaka exposed to PCB126. KC-400 exposure suppressed 15 genes and induced 266 genes in medaka brain. Among these genes, the expression levels of 7 and 188 genes were commonly down- or up-regulated, respectively in both treatment groups. On the other hand, 31 gene expressions were significantly different between PCB126 and KC-400 treatment groups, and three out of 31 genes were received opposite effects. In addition, the microarray data showed that thyroid hormone-responsive genes were up-regulated by PCB-exposure, which may imply that PCBs or their metabolites mimic thyroid hormone effects in the brain of PCB-exposed medaka.     

Levels of endocrine disrupting compounds in South China Sea

The occurrence of estrogens in the aquatic environment has become a major concern worldwide because of their strong endocrine disrupting potency. In this study, concentrations of four estrogenic compounds, estrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol (EE2), estriol (E3) were determined with liquid chromatography-tandem mass spectrometry analyses in surface water from South China Sea, and distributions and potential risks of their estrogenic activity were assessed. The estrogenic compounds E1, E2 and E3 were detected in most of the samples, with their concentrations up to 11.16, 3.71 and 21.63 ng L⁻¹. However, EE2 was only detected in 3 samples. Causality analysis, EEQ values from chemical analysis identified E2 as the main responsible compounds. Based on the EEQ values in the surface water, high estrogenic risks were in the coastal water, and low estrogenic risks in the open sea.     

Occurrence of estrogen-like substances in the marine environment of the Northern Mediterranean Sea

Evidence has been presented that a number of environmental agents perturbs the function of the sex hormone signalling pathways in marine animals.

In this work the estrogenic/antiestrogenic activity of coastal marine water samples from five sites of the Northern Mediterranean Sea was assayed using a recombinant Saccharomyces cerevisiae yeast strain (RMY326 ER-ERE) transfected with the human estrogen receptor . Extraction of potential EDCs from seawater was performed in columns packed with XAD-2 resins.

The estrogenic activity was calculated as percentage of the activity of 17β-estradiol (10 nM) and it ranged from 4.8% and 59.03%, significantly depending on sampling site (p = 0.0013) and season (p < 0.05). Antagonistic activity of extracts was also detected and the percentage of inhibition of estradiol-dependent β-galactosidase induction ranged to 52.8%. These results point the reliability of the yeast assay as a first level screening test to assess the quality of aquatic environments.     

Effects of 17β-estradiol on the reproduction of Java-medaka (Oryzias javanicus), a new test fish species

17β-estradiol (E2) is a female hormone which is known to be one of the strongest estrogenic chemicals in the environment. The present study investigated the effects of E2 on the reproduction of the estuarine fish, Java-medaka (Oryzias javanicus). Starting from the embryonic stages, Java-medaka was exposed for 6 months to 9.5, 16, 68, 159 and 243 ng/l of E2. The fecundity of Java-medaka exposed to E2 levels >16 ng/l was significantly lower than that of the control. The appearance of secondary sexual characteristics seemed to be inhibited by exposure to 159 and 243 ng/l E2. Vitellogenin concentrations in the liver of male fish exposed to 68, 159 and 243 ng/l were significantly higher than that of the control. Thirty-three and sixty percent of male fishes exposed to 159 and 243 ng/l, respectively, had testis-ova. These results suggest that reproduction in the Java-medaka, especially the male fish, could be affected by exposure to E2 concentrations greater than 16 ng/l.     

Differential developmental toxicity of naphthoic acid isomers in medaka (Oryzias latipes) embryos

Polycyclic aromatic hydrocarbons (PAHs) are widespread persistent pollutants that readily undergo biotic and abiotic conversion to numerous transformation products in rivers, lakes and estuarine sediments. Here we characterize the developmental toxicity of four PAH transformation products each structural isomers of hydroxynaphthoic acid: 1H2NA, 2H1NA, 2H3NA, and 6H2NA. Medaka fish (Oryzias latipes) embryos and eleutheroembryos were used to determine toxicity. A 96-well micro-plate format was used to establish a robust, statistically significant platform for assessment of early life stages. Individual naphthoic acid isomers demonstrated a rank order of toxicity with 1H2NA > 2H1NA > 2H3NA > 6H2NA being more toxic. Abnormalities of circulatory system were most pronounced including pericardial edema and tube heart. To determine if HNA isomers were AhR ligands, spatial-temporal expression and activity of CYP1A was measured via in vivo EROD assessments. qPCR measurement of CYP1A induction proved different between isomers dosed at respective concentrations affecting 50% of exposed individuals (EC50s). In vitro, all ANH isomers transactivated mouse AhR using a medaka CYP1A promoter specific reporter assay. Circulatory abnormalities followed P450 induction and response was consistent with PAH toxicity. A 96-well micro-plates proved suitable as exposure chambers and provided statistically sound evaluations as well as efficient toxicity screens. Our results demonstrate the use of medaka embryos for toxicity analysis thereby achieving REACH objectives for the reduction of adult animal testing in toxicity evaluations.     

Estrogen equivalent concentration of individual isomer-specific 4-nonylphenol in Ariake sea water, Japan

Concentrations of 4-nonylphenol (NP) were determined by isomer-specific quantification of individual NP isomers based on relative response factor (RRF) quantification with GC–MS in combination with steam distillation extraction. Concentrations of NP in the Ariake Sea decreased with distance from the river mouth (St.A; 49 ng NP/l) to offshore areas (St.C; 11 ng NP/l). Even the least concentration in water from St.C in Ariake Sea was sufficient to have adverse effects on barnacles. The isomers, NP1–NP14 were separated by GC–PFC and identified structurally with NMR. The isomers varied in estrogenic activity with NP7 exhibiting the greatest estrogenic activity with a potency that was approximately 1.9 × 10⁻³ that of 17β-estradiol (E2) in recombinant yeast screen system. The coefficient of variation (CV) of NP isomer’s concentrations among three samples at St.A, B and C were 4–75%. This suggests that NP isomers might be independently degraded in aquatic environmental samples. The predicted estrogenic activity of measured concentrations of NP in Ariake Sea was 2.7–3.0-fold greater than the measured estrogen agonist activity.     

A numerical method to compute Euler potentials for non dipolar magnetic fields

The magnetospheric magnetic field may be conveniently described by two scalar functions (α, β), known as the Euler potentials. They are not uniquely defined, and they may be difficult to derive for configuration more complex than a simple dipole. We propose here a simple numerical method to compute one possible pair (α, β). In magnetospheric regions of closed field lines, α can be chosen as a function of the tube volume of unit magnetic flux. The method can be applied to a wide class of magnetic fields which describe the magnetospheric domain of closed field lines and the conjugated ionosphere. Here, it is used with the T87 Tsyganenko model. The results coincide with the dipolar potentials at close distances from the Earth. At larger distances, they display an increasing distortion with the radial distance (or the invariant latitude in the ionosphere) and the magnetic activity. In the magnetosphere, the contours of α and β are stretched towards the nightside. In the ionosphere, they also extend towards the nightside and present major distortions in a narrow ring at the polar cap boundary, which mapsdistant boundary layers in the magnetosphere.     

The North-South Asymmetry in the Green Corona

The north-south asymmetry of the Fe XIV 530.3 nm coronal emission line (the green corona) over cycle 22 was investigated. The green corona line brightness was dominant in the southern hemisphere during cycle 22 (A = –0.07), except for short periods of the ascending phase of the activity cycle. The asymmetry of the semi-annual mean over the period 1940 – 1996 was also studied. The asymmetry, during these years, reached its maximum in 1962 – 1966, and then decreased. Important periodicities of the asymmetry in cycle 22, e.g., 158 and 350 days, 2.39 years were found. Similar periodicities were also detected in the years 1940 – 1996. An FFT analysis was used to detect these periodicities.     

Cosmic factors influence on the inter-annual variations of the green 557.7 Nm line and red 630.0 Nm line nightglow intensities and their possible coupling with cloud covering at Abastumani (41.75°N, 42.82°E)

We studied the inter-annual distributions of the nightglow intensities of the thermosphere atomic oxygen red 630.0 nm and green 557.7 nm lines observed from Abastumani during cloudless nights, the planetary geomagnetic Ap index, solar F_10.7, and galactic cosmic rays (GCRs) flux. It is demonstrated that: on magnetically weakly disturbed/ quiet conditions (Ap < 12) in equinoctial months the red line intensities are minimal, while those of the green line are maximal; the red line intensity increases in May-July and is comparatively low in June, where, unlike most mid-latitude regions, the green line intensity is maximal. The red and green line intensities increase with growing solar activity but their behaviors stay the same, which is considered as a possible regional manifestation of lower and upper atmosphere vertical coupling. It was also detected that, for cloudless nights in June, the number of magnetically disturbed day-nights is maximal and the decrease of the GCRs flux is the biggest during a year.     

Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTα gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTα gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTα gene as rm-GSTα. The cDNA of the rm-GSTα gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTα was subcloned into a 6 × His-tagged pCR^®T7 TOPO TA expression vector to produce the recombinant 6 × His-tagged rm-GST protein. This will be used in future to raise an rm-GSTα antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 μg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTα mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTα gene as well as its potential use in relation to environmental pollution.     

Alteration of gene expression profiles in the brain of Japanese medaka (Oryzias latipes) exposed to KC-400 or PCB126

Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals and possibly alter animal behavior. We previously reported that PCB-exposure induced abnormal schooling behavior in Japanese medaka (Oryzias latipes). This abnormal behavior might be caused by the functional alteration of central or terminal nervous system. To understand the mechanism(s) of behavioral change by PCB-exposure, we analyzed the gene expression profiles in the brain of medaka exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) or a PCB mixture (Kanechlor-400: KC-400) using a cDNA microarray that we constructed. Twelve FLF-II strain medaka (six individuals per treatment) were dietary exposed to PCB126 (0.01 μg/g b.w./day) or KC-400 (1 μg/g b.w./day) for three weeks. For the control, six fish were fed a control diet. After the exposure period, fish were dissected, and the brain samples were collected. The samples from control fish were pooled and used as a common reference in the microarray experiment. Microarray data were normalized by the LOWESS method, and we screened the genes whose expression levels were altered more than 1.5-fold. Gene expression profiling showed 97 down-regulated and 379 up-regulated genes in the brain of medaka exposed to PCB126. KC-400 exposure suppressed 15 genes and induced 266 genes in medaka brain. Among these genes, the expression levels of 7 and 188 genes were commonly down- or up-regulated, respectively in both treatment groups. On the other hand, 31 gene expressions were significantly different between PCB126 and KC-400 treatment groups, and three out of 31 genes were received opposite effects. In addition, the microarray data showed that thyroid hormone-responsive genes were up-regulated by PCB-exposure, which may imply that PCBs or their metabolites mimic thyroid hormone effects in the brain of PCB-exposed medaka.     

Profiles of nonylphenol isomers in surface waters from Sri Lanka

Isomer-specific concentrations of nonylphenol (NP) and their predicted estrogenic potency were investigated in Sri Lankan waters for the first time. The total concentration of 13 NP isomers ranged from 90 to 1835 ng/L, while the predicted estrogenic equivalent concentration ranged from 0.072 to 1.38 ng 17β-estradiol (E2)/L. Bire Lake, located in the central area of the commercial capital, Colombo, had the highest contamination among the studied locations. These data show that NP levels in Sri Lankan waters are well within the recently reported concentrations in other regions of the world. The spatial differences in NP concentrations suggest that NP contamination in Sri Lanka may be widespread, and comprehensive study is vital.     

Levels of estrogenic compounds in Xiamen Bay sediment, China

Concentrations of seven estrogenic compounds, i.e., estrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol(EE2), diethylstilbestrol (DES), nonylphenol (NP), octylphenol (OP) and bisphenol A (BPA), were determined in sediments and pore water from Xiamen Bay in China, and their distributions and potential risks in the Bay were assessed. Total estrogenic compounds concentrations varied from 49.20 to 1230.69 ng/g dw in sediments and from 102.33 to 4376.60 ng/L in pore water. The highest levels of these compounds were found at Yundang Lagoon. The results showed that estrogenic compounds in Xiamen Bay originated mainly from municipal wastewaters. Compared with other areas, Xiamen Bay was contaminated with high levels of estrogen hormones. This contamination poses a potential threat to benthic organisms. Although a good relationship (r = 0.94) was observed between the estrogenic compounds concentrations and total organic carbon (TOC) contents in sediments, which did not indicate that the sediment organic matter favors the accumulation of the detected estrogenic compounds.     

Carbon and oxygen isotope composition of shell carbonate of desert land snails

Variation in¹³C/¹²C and¹⁸O/¹⁶O ratios in the shell carbonate of several species of land snails was studied along a climatic gradient in semi-arid to arid areas in the southern Levant.¹³C was found to be enriched in snails from communities having plants with a C₄ photosynthetic pathway. Depleted δ¹³C values were found in areas with high mean annual rainfall, apparently due to higher input of metabolic CO₂ as a result of greater snail activity. Shell carbonate δ¹⁸O values show a weak relation to the δ¹⁸O values of rainwater. The shell δ¹⁸O values are enriched by 2–8‰ relative to isotopic equilibrium with environmental waters. Enrichment is suggested to result from metabolic effects on body water (with lower activity producing greater enrichment) but evaporation could also be a factor. Consistent differences in both¹³C and¹⁸O were found among species and may relate to the time when shell deposition occurs. As with most paleoenvironmental indicators, the application of shell isotopes is complicated by the multiplicity of controls of isotopic composition.     

The green oxygen line 5577 Å in the mediumE-F andF regions during the night

Summary In this paper the contribution of theF region to the green oxygen line 5577 Å glow is investigated. It is shown that at calm nocturnal conditions the ions' O ₂ ⁺ dissociative recombination leads to a simultaneous excitation of the red and green lines in this region. It has been found that the intensity of the green line in theF region is about one-third that of the red line. By this non-negligible component we can explain some unexplained phenomena in the behaviour of the green line during the night.     

Water–Rock interactions in the basement beneath Long Valley Caldera: an oxygen isotope study of the Long Valley Exploratory Well drill cores

The Long Valley Exploratory Well, at the center of the Resurgent Dome of Long Valley caldera, penetrated pre-caldera basement rocks at a depth of 2101.72–2313.0 m, beneath the caldera-forming Bishop Tuff and post-caldera Early Rhyolite. The basement rocks contain prominent quartzites, with ubiquitous milky white quartz veins (with minor calcite and pyrite) and fractures of varied orientation and geometry. The other members of the basement sequence are very fine-grained quartz-rich graphitic pelites with calcite veins, spotted hornfels, and shallow intrusive rocks. Previous studies established the presence of a post-caldera, paleohydrothermal system (500–100 ka) to a depth of 2000 m that affected the Bishop Tuff and a recent (40 ka to present) hydrothermal system at shallow depth (<1 km). The deeper extent of these hydrothermal activities is established in this paper by a detailed oxygen isotope analysis of the drill core samples. 238 analyses of δ¹⁸O in 50 quartz veins within the 163.57 m depth interval of basement rocks reveal extreme heterogeneity in δ¹⁸O values (8–19.5‰). Majorities of the 84 bulk analyses of quartzites show variation of δ¹⁸O within a narrow range of 14–16‰. However, certain samples of these quartzites near the contacts with veins and fractures exhibit sharp drops in δ¹⁸O. The interbedded pelitic rocks and spotted hornfels have whole-rock δ¹⁸O ranging from 2.2 to 11.8‰. Clear, euhedral vuggy quartz that partially fills earlier open fractures in both the quartzites and quartz veins, has distinctive δ¹⁸O, ranging between −3.2 and +8.4‰. Low values of δ¹⁸O are also found in the hydrothermal minerals and whole rocks adjacent to the thin veins, clearly indicating infiltration of meteoric water. Three distinct observed patterns of fractionation in δ¹⁸O between veins and host quartzites are analyzed with the principles of mass balance, equilibrium oxygen isotope fractionation in closed system, and kinetically controlled oxygen isotope exchange in an open system. This analysis suggests that the early quartz veins formed due to a magmatic-hydrothermal activity with no influx of external water once the system comprising the sedimentary envelope and a magmatic-hydrothermal fluid phase became closed. Two-stage isotopic exchange processes caused fractionation in the δ values that originally formed arrays with slope 1 in a δ_{vein quartz}–δ_{host quartzite} space. Another array in the same space, with near zero slope was also formed due to variation in temperature, initial isotopic compositions of the quartzite sequence and the fluid phase. Variation in temperature was mostly in the range of 300–400°C giving Δ (=δ_{vein quartz}–δ_{host quartzite})≈−2.8 to +2.8. The δ¹⁸O of the fluid could range from −5 to +10; however a narrower range of +5 to +10 can explain the data. This episode of hydrothermal activity could take place either as a single pulse or in multiple pulses but each as a closed system. A later, fracture-controlled, meteoric water (δ¹⁸O−0.46 to −12.13) flow and interaction (at 250°C) is interpreted from the analysis of δ¹⁸O values of the coexisting quartz and calcite pairs and existence of markedly ¹⁸O-depleted pelitic horizons interbedded with ¹⁸O-enriched quartzite layers. Thus, the interpreted earlier magmatic-hydrothermal activity was overprinted by a later meteoric-hydrothermal activity that resulted in steep arrays of δ¹⁸O values in the δ_{vein quartz}–δ_{host quartzite} space. Calculations show that the likely life span of the post-caldera, hydrothermal activity in the depth range of 2.1–2.3 km beneath Long Valley was 0.08–0.12 Ma. Diffusive ±advective transport of oxygen isotopes from fracture-channelized meteoric water to nearly impermeable wall rocks caused a lowering of δ¹⁸O values in the quartz over short distances and in calcites over greater distances. Thus, the hydrothermal activity appears pervasive even though the meteoric water flow was primarily controlled by fractures.     

Dayside isotropic precipitation of energetic protons

Recently it has been shown that isotropic precipitation of energetic protons on the nightside is caused by a non-adiabatic effect, namely pitch-angle scattering of protons in curved magnetic field lines of the tail current sheet. Here we address the origin of isotropic proton precipitation on the dayside. Computations of proton scattering regions in the magnetopheric models T87, T89 and T95 reveal two regions which contribute to the isotropic precipitation. The first is the region of weak magnetic field in the outer cusp which provides the 1–2° wide isotropic precipitation on closed field lines in a 2–3 hour wide MLT sector centered on noon. A second zone is formed by the scattering on the closed field lines which cross the nightside equatorial region near the magnetopause which provides isotropic precipitation starting 1.5–2 h MLT from noon and which joins smoothly the precipitation coming from the tail current sheet. We also analyzed the isotropic proton precipitation using observations of NOAA low altitude polar spacecraft. We find that isotropic precipitation of >30 to > 80 keV protons continues around noon forming the continuous oval-shaped region of isotropic precipitation. Part of this region lies on open field lines in the region of cusp-like or mantle precipitation, its equatorward part is observed on closed field lines. Near noon it extends 1–2° below the sharp boundary of solar electron fluxes (proxy of the open/closed field line boundary) and equatorward of the cusp-like auroral precipitation. The observed energy dispersion of its equatorward boundary (isotropic boundary) agrees with model predictions of expected particle scattering in the regions of weak and highly curved magnetic field. We also found some disagreement with model computations. We did not observe the predicted split of the isotropic precipitation region into separate nightside and dayside isotropic zones. Also, the oval-like shape of the isotropic boundary has a symmetry line in 10–12 MLT sector, which with increasing activity rotates toward dawn while the latitude of isotropic boundary is decreasing. Our conclusion is that for both dayside and nightside the isotropic boundary location is basically controlled by the magnetospheric magnetic field, and therefore the isotropic boundaries can be used as a tool to probe the magnetospheric configuration in different external conditions and at different activity levels.